Enrichment and cultivation of prokaryotes associated with the sulphate-methane transition zone of diffusion-controlled sediments of Aarhus Bay, Denmark, under heterotrophic conditions

The prokaryotic activity, diversity and culturability of diffusion-controlled Aarhus Bay sediments, including the sulphate-methane transition zone (SMTZ), were determined using a combination of geochemical, molecular (16S rRNA and mcrA genes) and cultivation techniques. The SMTZ had elevated sulphate reduction and anaerobic oxidation of methane, and enhanced cell numbers, but no active methanogenesis. The prokaryotic population was similar to that in other SMTZs, with Deltaproteobacteria, Gammaproteobacteria, JS1, Planctomycetes, Chloroflexi, ANME-1, MBG-D and MCG. Many of these groups were maintained in a heterotrophic (10mM glucose, acetate), sediment slurry with periodic low sulphate and acetate additions (similar to 2mM). Other prokaryotes were also enriched including methanogens, Firmicutes, Bacteroidetes, Synergistetes and TM6. This slurry was then inoculated into a matrix of substrate and sulphate concentrations for further selective enrichment. The results demonstrated that important SMTZ bacteria can be maintained in a long-term, anaerobic culture under specific conditions. For example, JS1 grew in a mixed culture with acetate or acetate/glucose plus sulphate. Chloroflexi occurred in a mixed culture, including in the presence of acetate, which had previously not been shown to be a Chloroflexi subphylum I substrate, and was more dominant in a medium with seawater salt concentrations. In contrast, archaeal diversity was reduced and limited to the orders Methanosarcinales and Methanomicrobiales. These results provide information about the physiology of a range of SMTZ prokaryotes and shows that many can be maintained and enriched under heterotrophic conditions, including those with few or no cultivated representatives.